3D Molecular Tracking in Live Cells with Simultaneous Time-Resolved Spectroscopy

Abstract

We report a confocal feedback method for tracking the motion of individual quantum dot labeled proteins as they move in three dimensions inside of cells that has sub-nanosecond temporal resolution. 3D tracking molecular is possible for tens of microns in X, Y, and Z, meaning tracking can occur throughout the entire cell volume volume for many cell lines. The sub-nanosecond temporal resolution enables time-resolved spectroscopies (e.g., fluorescence lifetime measurements or fluorescence correlation spectroscopy) to be made on the molecules as they are being tracked. In particular, recording of the arrival times of individual photons enabled, for the first time, photon pair correlation measurements showing fluorescence photon anti-bunching of individual QD labeled proteins in live cells. The power of this new technology is further illustrated through tracking of individual QD-labeled IgE receptor complexes on rat mast cells, revealing three-dimensional nano-scale topology of the cell membrane as individual receptors navigate hills and valleys of a dynamically changing plasma membrane landscape. In addition to mapping out cell surface topology, IgE-Fc{epsilon}RI signaling clusters were also captured in the act of ligand-mediated endocytosis and tracked during rapid (∼950 nm/s) vesicular transit through the cell.